Recombinant Chicken Uncharacterized protein C11orf73 homolog (RCJMB04_13p7)

What is the function of C11orf73 homolog in vertebrate systems?

The C11orf73 homolog (also referred to as C11ORF73 in some literature) appears to play a significant role in cellular processes related to lysosomal function and mTOR signaling. Research indicates that wild type C11ORF73 constructs have the ability to promote lysosome-related S6 and 4E-BP1 phosphorylation, which is essential for cellular differentiation processes . In neural cells specifically, this protein may contribute to oligodendrocyte differentiation and myelination through its effects on mTOR signaling pathways .

Methodologically, researchers investigating this protein's function typically employ:

• Plasmid constructions encoding the full-length gene

• Transfection of appropriate cell lines (such as FBD-102b oligodendroglial cells)

• Assessment of downstream signaling effects through phosphorylation assays

• Cellular localization studies using fluorescent tagging

How should researchers design experiments to express and purify recombinant chicken C11orf73 homolog?

When expressing and purifying this protein, researchers should consider the following methodological approach:

• Gene amplification and vector construction:

  • Amplify the chicken c11orf73 homolog gene (RCJMB04_13p7) using Gflex DNA polymerase or similar high-fidelity enzyme

  • Ligate the amplified fragments with an appropriate expression vector (e.g., pEGFP-C1 for GFP-fusion proteins)

  • Confirm all DNA sequences by sequencing

• Expression system selection:

  • For mammalian expression: Use COS-7 cells which demonstrate high transfection efficiency (approximately 75%)

  • For avian-specific studies: Consider chicken cell lines or avian-origin cells

  • Transfect using methods like ScreenFect A or ScreenFect A Plus according to manufacturer protocols

• Purification strategy:

  • For tagged proteins: Use sequential affinity chromatography (e.g., with FLAG-tag and His-tag in tandem)

  • Employ cell lysis with appropriate buffers (e.g., RIPA Buffer: 50 mM sodium phosphate, pH 7.4, 1 mM MgCl₂, 150 mM NaCl, 1% Nonidet P-40, 0.8% deoxycholate)

  • Include protease inhibitors to prevent degradation

What expression patterns and tissue distribution have been observed for C11orf73 homolog proteins?

While specific expression data for the chicken homolog is limited in the provided search results, related research on mammalian C11orf73 suggests:

• Expression in neural tissues, particularly in oligodendrocytes

• Potential expression in multiple tissues based on its role in fundamental cellular processes

• Subcellular localization primarily in lysosomes for wild-type protein

For researchers studying tissue distribution:

• Consider using RT-PCR or RNA-Seq across multiple chicken tissues to establish expression profiles

• Employ immunohistochemistry with specific antibodies against the chicken homolog

• Develop tissue-specific conditional knockout models to evaluate function in particular tissues

How can researchers effectively analyze mutations in chicken C11orf73 homolog and their functional consequences?

Based on studies of mammalian C11orf73, mutations can significantly alter protein function. To analyze mutations in the chicken homolog:

Site-directed mutagenesis methodology:

• Use the wild-type chicken C11orf73 homolog as a template

• Design primers incorporating desired mutations (based on conservation with human mutations like C4S or V54L)

• Employ a site-directed mutagenesis kit according to manufacturer's instructions

• Confirm all mutations by sequencing

Functional analysis approach:

• Compare cellular localization of wild-type and mutant proteins using fluorescent tags

• Assess impact on lysosomal function through markers of lysosomal activity

• Evaluate downstream signaling effects, particularly mTOR pathway components

• Quantify protein-protein interactions through co-immunoprecipitation

Researchers should pay particular attention to mutations equivalent to human C4S (Cys4-to-Ser) and V54L (Val54-to-Leu), as these have demonstrated significant phenotypic effects in mammalian studies .

What proteomics approaches are most effective for identifying interaction partners of chicken C11orf73 homolog?

Mass spectrometry-based identification of protein interaction partners provides crucial insights into protein function. Based on methodologies used for similar proteins:

Recommended proteomics workflow:

• Sample preparation:

  • Express tagged C11orf73 homolog in appropriate cell lines

  • Perform protein complex purification through sequential affinity chromatography

  • Process samples using TCA precipitation for concentration

• Protein digestion and peptide preparation:

  • Digest purified complexes with Trypsin Gold (1 U/mL overnight)

  • Treat with iodoacetamide to prevent disulfide bond re-formation

  • Separate trypsin-degraded peptides by L-column 2 ODS with gradient acetonitrile (0-60%) in 0.1M trifluoroacetic acid

• Mass spectrometry analysis:

  • Employ LC-MS/MS for peptide detection and identification

  • Consider techniques like GeLC-MS/MS with repetitive LC-MS/MS analysis for enhanced protein identification

  • Use comparative proteomics between wild-type and mutant proteins to identify differential binding partners

The table below summarizes comparative proteomics approaches that can be applied to C11orf73 homolog research:

Data adapted from comparative proteomics studies

How can researchers design knockdown or knockout experiments to study C11orf73 homolog function in chicken models?

For functional studies of C11orf73 homolog, gene silencing provides valuable insights:

RNA interference (RNAi) approach:

• Design siRNAs targeting conserved regions of chicken C11orf73 homolog

• Transfect primary chicken cells or chicken cell lines using optimized protocols

• Confirm knockdown efficiency by qRT-PCR and Western blot

• Assess phenotypic changes in cellular processes including lysosomal function and mTOR signaling

CRISPR-Cas9 methodology for knockout studies:

• Design guide RNAs targeting early exons of chicken C11orf73

• Validate guide RNA efficiency in chicken cell lines

• For in vivo studies, consider embryonic manipulation techniques specific to avian models

• Verify knockout by sequencing and protein expression analysis

Phenotypic assessment protocol:

• Evaluate changes in cellular morphology and viability (use trypan blue exclusion test)

• Analyze lysosomal function using specific markers and activity assays

• Assess mTOR pathway components through phosphorylation status

• Perform cellular differentiation assays if working with relevant cell types

What methods are most appropriate for studying the impact of C11orf73 homolog on cellular signaling pathways?

Based on mammalian studies, C11orf73 appears to influence mTOR signaling pathways. To investigate this in the chicken homolog:

Signaling pathway analysis methodology:

• Establish stable cell lines expressing wild-type or mutant chicken C11orf73 homolog

• Use G418 selection (0.1250 mg/mL) to isolate stable transfectants over approximately 14 days

• Compare G418-resistant colonies for phenotypic differences against control cells

Specific signaling assays:

• mTOR pathway components:

  • Evaluate phosphorylation status of S6 and 4E-BP1 proteins by Western blot

  • Quantify changes under various cellular conditions (starvation, stimulation)

  • Compare wild-type vs. mutant protein effects on signaling dynamics

• Interaction with cytoskeletal proteins:

  • Assess binding to proteins like Filamin A, which anchors transmembrane proteins to actin cytoskeleton

  • Use co-immunoprecipitation followed by Western blot or MS analysis

  • Perform functional assays to determine physiological relevance of interactions

What are the key considerations for designing controlled experiments with chicken C11orf73 homolog?

When designing experiments for this relatively uncharacterized protein, researchers should implement:

Experimental design framework:

• Define clear research questions and hypotheses

  • Formulate specific questions about protein function, interactions, or effects

  • Develop testable hypotheses based on mammalian C11orf73 literature

• Select appropriate controls

  • Include empty vector controls for expression studies

  • Use scrambled siRNA or non-targeting CRISPR guides for knockdown/knockout studies

  • Consider species-specific positive controls for signaling pathway studies

• Variable management

  • Control for transfection efficiency variations between experiments

  • Maintain consistent cell passage numbers

  • Standardize expression levels when comparing wild-type and mutant proteins

• Replication strategy

  • Perform biological replicates (n≥3) for all key experiments

  • Include technical replicates for methods with high variability

  • Consider power analysis to determine appropriate sample sizes

How should researchers analyze and interpret contradictory data in C11orf73 homolog studies?

When faced with contradictory results:

Data contradiction resolution methodology:

• Systematic verification of experimental conditions

  • Assess whether differences in cell types, expression levels, or assay conditions could explain discrepancies

  • Verify reagent quality and specificity, particularly antibodies

• Sequential hypothesis refinement

  • Develop alternative hypotheses that could explain contradictory findings

  • Design critical experiments to distinguish between competing hypotheses

• Analytical approaches for reconciling discrepancies

  • Perform meta-analysis of multiple experimental replicates

  • Consider whether contradictions reflect genuine biological variability or technical artifacts

  • Evaluate whether conflicting observations might represent context-dependent protein functions

• Statistical considerations

  • Apply appropriate statistical tests based on data distribution

  • Consider non-parametric methods for data that violates normality assumptions

  • Report effect sizes alongside p-values to assess biological significance

What statistical approaches are most appropriate for analyzing data from C11orf73 homolog functional studies?

Statistical analysis framework:

• For protein expression and localization studies:

  • Quantify fluorescence intensity using image analysis software

  • Compare distributions using appropriate parametric or non-parametric tests

  • Consider spatial statistics for co-localization analyses

• For signaling pathway analyses:

  • Use repeated measures ANOVA for time-course experiments

  • Apply multiple comparison corrections (e.g., Bonferroni, Tukey's HSD) when testing multiple conditions

  • Employ multivariate analysis for complex multi-pathway datasets

• For protein-protein interaction studies:

  • Calculate enrichment ratios compared to appropriate controls

  • Apply statistical filters (p-value, fold change) to identify high-confidence interactors

  • Consider Bayesian approaches for integration of multiple datasets

• Sample size considerations:

  • Perform power analysis to determine appropriate sample sizes for detecting biologically meaningful effects

  • Report confidence intervals alongside point estimates

  • Consider hierarchical or mixed-effects models for nested experimental designs

How does chicken C11orf73 homolog compare structurally and functionally to mammalian orthologs?

For researchers interested in comparative studies:

Comparative analysis methodology:

• Sequence comparison approach:

  • Perform multiple sequence alignment of C11orf73 homologs across species

  • Identify conserved domains and critical residues

  • Focus particular attention on regions corresponding to known functional mutations (e.g., C4S, V54L)

• Structure prediction methodology:

  • Generate structural models using homology modeling

  • Validate models through energy minimization and Ramachandran plot analysis

  • Identify potential functional sites through structural analysis

• Functional conservation assessment:

  • Test whether chicken C11orf73 can complement mutations in mammalian cells

  • Compare subcellular localization patterns across species

  • Analyze conservation of protein-protein interactions

The table below illustrates a hypothetical comparison of key features between chicken and human C11orf73:

What approach should be used to study evolutionary conservation of C11orf73 homolog function across species?

Evolutionary analysis methodology:

• Phylogenetic analysis:

  • Construct phylogenetic trees using maximum likelihood or Bayesian methods

  • Analyze rates of evolution in different protein domains

  • Identify signatures of selection acting on specific regions

• Functional complementation studies:

  • Express chicken C11orf73 in mammalian cells with endogenous C11orf73 knockdown

  • Assess rescue of phenotypes associated with loss of mammalian protein

  • Compare wild-type and mutant forms in cross-species complementation

• Synteny analysis:

  • Evaluate conservation of genomic context around C11orf73 locus

  • Identify conserved regulatory elements

  • Assess co-evolution with interacting partners

How can researchers optimize mass spectrometry protocols for characterizing chicken C11orf73 homolog and its interactions?

Mass spectrometry (MS) provides critical insights into protein interactions and modifications:

MS optimization methodology:

• Sample preparation refinement:

  • Employ sequential affinity chromatography for purifying protein complexes

  • Consider crosslinking approaches to capture transient interactions

  • Use TCA precipitation for sample concentration

• Digestion protocol optimization:

  • Digest with Trypsin Gold (1 U/mL) overnight

  • Treat with iodoacetamide to prevent disulfide bond re-formation

  • Separate peptides using gradient elution with acetonitrile (0-60%)

• MS data acquisition strategy:

  • Compare different MS approaches (e.g., 2-D vs. 3-D methods)

  • Implement filtering criteria: X corr ≥ 1.8 (+1); 2.1 (+2); 3.25 (+3), Δ C n ≥ 0.05

  • Consider alternative approaches (GeLC-MS/MS) for improved coverage

• Data analysis considerations:

  • Apply appropriate filtering to identify high-confidence peptides

  • Compare results across biological replicates

  • Validate key interactions through orthogonal methods (co-IP, proximity labeling)

What cell-based assays are most informative for characterizing chicken C11orf73 homolog function?

Based on knowledge of mammalian C11orf73:

Cell-based functional assay methodology:

• Lysosomal function assays:

  • Measure lysosomal enzyme activity in cells expressing wild-type vs. mutant protein

  • Assess lysosomal pH using appropriate indicators

  • Evaluate autophagy flux through LC3 conversion and p62 degradation

• mTOR signaling pathway analysis:

  • Quantify phosphorylation of S6 and 4E-BP1 through Western blot or ELISA

  • Measure effects on protein synthesis using puromycin incorporation

  • Assess mTOR localization and activity under various cellular conditions

• Cell viability and morphology assessments:

  • Monitor cell viability using trypan blue exclusion test (<5% incorporation)

  • Evaluate cellular morphology through microscopy

  • Assess proliferation rates in cells with wild-type vs. mutant protein

How might findings from chicken C11orf73 homolog studies translate to understanding human disease mechanisms?

Research on C11orf73 homologs has implications for understanding human diseases:

Translational research methodology:

• Disease model development:

  • Establish chicken models with mutations corresponding to human disease variants

  • Validate phenotypes that recapitulate aspects of human disorders

  • Use comparative analyses to identify conserved disease mechanisms

• Therapeutic target evaluation:

  • Assess whether interventions targeting C11orf73-related pathways show efficacy

  • Evaluate effects on downstream signaling in disease models

  • Consider species differences when extrapolating findings to human contexts

• Biomarker identification approach:

  • Identify changes in protein expression or modification associated with dysfunction

  • Evaluate whether such changes could serve as disease biomarkers

  • Validate findings across multiple model systems

The importance of careful methodological approach in translational studies is underscored by observations in human disease research, where infantile leukoencephalopathy-associated mutations in C11ORF73 demonstrate specific effects on lysosomal function and mTOR signaling .

What methodological approach is recommended for investigating the role of C11orf73 homolog in developmental processes?

For developmental studies:

Developmental biology research methodology:

• Temporal expression analysis:

  • Profile C11orf73 homolog expression across developmental stages

  • Use RT-qPCR, RNA-Seq, or in situ hybridization for spatiotemporal patterns

  • Compare with known developmental markers

• Conditional manipulation approach:

  • Develop systems for stage-specific or tissue-specific manipulation

  • Consider electroporation techniques for chicken embryo studies

  • Evaluate effects on tissue development and differentiation

• Signaling pathway integration:

  • Assess how C11orf73 homolog interacts with known developmental pathways

  • Focus particularly on mTOR signaling components

  • Investigate potential roles in cell fate determination and differentiation